Although significant advances in treating the nearly one million Americans infected with HIV-1 have been achieved with the use of antiviral nucleoside reverse transcriptase inhibitors (NRTIs), NRTI-associated mitochondrial toxicities and side effects pose serious risks of non-HIV related morbidity and challenge patient adherence to the daily drug regimens necessary to maintain reduced viral loads and prevent viral transmission. Prominent NRTI-associated toxicities arise from viral versus host polymerase selectivity, wherein NRTIs not only function to halt viral replication by chain termination for reverse transcriptase (RT) but can also serve as substrates for host polymerases and result in severe mitochondrial dysfunction. The recent discovery of a novel human primase-polymerase, PrimPol, operating in the mitochondria and possessing the ability to incorporate five NRTI substrates suggests a novel mechanism of antiviral toxicity that has yet to be explored. This proposal seeks to investigate the molecular, structural, and cellular mechanisms of PrimPol's catalytic activities and evaluate its potential as a mechanism of mitochondrial NRTI-associated toxicity. First, pre- steady-state and steady-state kinetics will be used to determine the incorporation efficiency of known PrimPol NRTI substrates and evaluated in the context of incorporation efficiencies for the natural correct and incorrect deoxynucleotide substrates to assess the propensity for toxicity. Second, a three-dimensional crystal structure of PrimPol will be obtained to aide in understanding the structural features that determine functionality and substrate specificity. Importantly, this will facilitate detailed structural comparisons with HIV-1 RT and the human mitochondrial DNA polymerase-? to aide in exploiting the differences therein for rational drug design of novel antivirals with increased selectivity and minimal toxicities. Thid, cellular studies elucidating the role of PrimPol in mitochondria function and in the maintenance of the mitochondrial genome will identify the downstream effects of NRTI inhibition of PrimPol. The mechanistic, structural, and cellular studies of PrimPol proposed are critical for elucidating a novel mechanism of NRTI-associated toxicities and for assessing the safety profile of FDA-approved NRTIs and those in development. As there is no cure for HIV-1 infection, the life-long administration of NRTIs is necessary and addressing the mechanisms of toxicity is critical to the effective care of HIV-1 infected patients.